Method for manufacture of polyphenols by using grape seeds as starting material

ABSTRACT

To provide a method for the manufacture of polyphenols that enables industrial manufacture of polyphenols containing water-soluble proanthocyanidins from grape seeds.

CROSS REFERENCE

This nonprovisional application claims the benefit of JapaneseApplication No.: 2005-002002, filed on Jan. 07, 2005 in Japan, which isincorporated herein by reference in its entirety and to which priorityis claimed.

FIELD OF THE INVENTION

The present invention relates to a method for the manufacture ofpolyphenols by using grape seeds as a starting material, this methoddesigned to obtain polyphenols comprising proanthocyanidins converted toa water soluble form.

BACKGROUND OF THE INVENTION

Because polyphenols contained in plants produce an antioxidation effect,they have been used in a large variety of food products, drugs, andcosmetics. Proanthocyanidins are polyphenols that produce an especiallystrong antioxidation effect. As described in Patent Reference 1,proanthocyanidins are polyphenols contained in a large amount in plantssuch as grapes, apples, persimmon, and pine, and because of theirespecially strong antioxidation effect, they are used for a variety ofapplications described, for example, in Para. Nos. 0031-0034 of PatentReference 1.

[Patent Reference 1]

Japanese Patent Application Laid-open No. 2001-158739.

Problems Addressed by the Invention

However, in the form in which proanthocyanidins are most generallypresent in the natural world, they are most often insoluble in water.For example, in grape berries, though proanthocyanidins are contained inpeel, flesh and seeds, they are all insoluble in water. For this reason,the field of application for proanthocyanidins tends to be relativelylimited. For example, when they are used in cosmetics, though they canbe contained in creams, they cannot be contained in cosmetic tonicscontaining no added alcohol. Furthermore, when used orally (food etc.),because water-insoluble proanthocyanidins are difficult to absorb, theuse thereof is avoided and other polyphenols that are readily soluble inwater are widely used despite their poor antioxidation effect.Therefore, though proanthocyanidins are examples of ingredients actuallycontained in food products and the like, as long as they arewater-insoluble proanthocyanidins, a sufficient antioxidation effectcannot be demonstrated. Some plants contain water-solubleproanthocyanidins, but they cannot ensure stable supply of large amountsof water-soluble proanthocyanidins, and there is need for industrialtechnology capable of guaranteeing such a supply.

The present invention focuses attention on the problems in theconventional technology described above. An object thereof is to providea method for the manufacture of polyphenols that enables industrialmanufacture of polyphenols containing water-soluble proanthocyanidinsfrom grape seeds.

[Means to Resolve the Problems]

In order to resolve the above-described problems, the invention of claim1 provides a method for the manufacture of polyphenois comprising adrying step of drying grape seeds after juice removal in which the grapeseeds are dried alone or with seed coat, a heating step of heating thegrape seeds dried in the drying, step in a temperature range above thetemperature optimum for germination and under heating conditions suchthat cause no thermal transformation of proteins present in the grapeseeds, a hydration step of hydrating the grape seeds simultaneously withthe heating step or after the heating step, and a germination inductionstep of germinating or germinating and differentiating the grape seeds,which were heated in the heating step and hydrated in the hydrationstep, in a temperature range optimum for germination, wherein apolyphenol comprising a proanthocyanidin converted to a water-solubleform is extracted from the grape seeds alter the germination inductionstep.

in addition to the features mentioned ahove, the present inventionfurther include the feature of an index of the degree of dryness in thedrying Step has a water content ratio of the grape seeds, and the targetvalue of the water content ratio is 9-12%.

Further the present invention also includes an index of water contentratio in the hydration step, wherein the target value of the watercontent ratio is 17% or more.

Still further, present invention, in addition to the features mentionedabove, in the heating step, temperature range above the temperatureoptimum for germination of the grape seeds is 50-70° C.

Employing the above-described features makes it possible to inducegermination or germination and differentiation of grape seeds, and toextract polyphenols comprising proanthocyanidins converted to a watersoluble form.

Usually, proanthocyanidins contained in grape seeds are insoluble inwater. Therefore, they have to be converted into water-solubleproanthocyanidins. For this purpose, the inventors, first, havesubjected the grape seeds after juice removal to drying in which thegrape seeds are dried alone or with seed coats. Owing to the drying, thecontent ratio of water in the grape seeds is decreased and a transitionis made to a type of dormant state.

The meaning of drying will be described below based on plant physiology.In the drying step, juice is removed and the internal seeds are exposedto air and dried naturally or forcibly, thereby differentiating thestarch present in the grape seeds to accumulated starch anddifferentiating the germination-related substances. The drying not onlyreleases free water of the seeds to the outside of the seed body, but atthe same time also enhances the release of the bonded water to theoutside of seed body by the differentiation of those substances of theseeds themselves by external stimulation, i.e., drying. For example,even when seeds that were directly taken from ripe grape berries areplanted in a temperature range optimum for germination, the germinationratio is very low (about 2-3% for the grapes). This is due to the factthat physiological conditions required for germination have not beenfully arranged. On the other hand, subjecting the grape seeds to thedrying step provides a necessary condition for germination. It does notmean that grapes that passed the dormant stage will necessarilydemonstrate rapid increase in germination ratio, as certain plants doafter passing the dormant stage. However, the germination ratio isdifficult to increase if the grape seeds have not passed at least thedrying step.

As drying advances, the grape seeds make a transition to a type ofdormant state. In the present invention, the grape seeds may be in adormant state or a state close to a dormant state (theoretically,differentiation to accumulated starch and differentiation ofgermination-related substances may be completed). The water contentratio may be an indicator of whether this state has been attained ornot. According to the idea of the present invention, the preferred watercontent ratio that is the target of drying is 9-12%, more preferably9-11%. The water content ratio of seeds present in grape berries isgenerally about 14%, regardless of the kind of grapes. Grape seeds donot have a water content ratio of more than 14%, unless they aregerminated and differentiated to a physiologically allowed limit (inother words, as long as they are alive).

Furthermore, because a certain time is required for the differentiationof grape seeds, from the standpoint of germination ratio increase, it ispreferred that the drying step be implemented slowly over many days(several weeks to several months) rather than within a short interval.

For example, dregs (the so-called juice pomace) remaining aftersqueezing out the juice for the fabrication of juice or making a whitewine or dregs (the so-called wine pomace) appearing in the process ofmaking a red wine can be used as means for naturally drying the seeds.If those dregs are open-air stored for a fixed period, dried, and usedas a starting material, then gradual drying is conducted, as describedhereinabove, and cost can be advantageously reduced. Furthermore,another effect that can be expected is that open-air storage causesdecay of seed coats, thereby facilitating subsequent separation of seedsand seed coats in the below-described washing process. A large electricfan or heater can be also considered as drying means for forcibledrying, but in this case, too, the germination ratio can be increased byallowing the seeds to stay after drying for several weeks to severalmonths, as described above, rather than making an immediate transitionto the next step.

Furthermore, after the above-described drying step, the inventors haveheated the grape seeds in a temperature range above the temperatureoptimum for germination and under heating conditions such that cause nothermal transformation of proteins present in the grape seeds.

The germination ratio has been planned to be greatly increased byintensely heating, the grape seeds that were made dormant by drying. Atthis stage, seed coats are preferable removed from the grape seeds, butthey may be also ich intact. Here, “the heating temperature optimum forgermination of grape seeds” is considered to be about 20-45° C., morepreferably 23-40° C. Furthermore, “the temperature range above theheating conditions causing no thermal transformation of proteins presentin the grape seeds” does not necessarily mean that a high temperature isexcluded. Because the grape seeds are covered with seed coats, heat doesnot directly penetrate into the seeds it means that the treatment can heconducted at a high temperature (for example 65-70° C.) causingtransformation of proteins, provided that the processing time is short.Conversely, long-teon heating can be employed if the temperature iscomparatively low (for example, about 50° C.). In some cases heating canbe conducted at a temperature of 100° C. or higher in an evaporationkettle or the like. The temperature and heating time are inverselyproportional to each other. However, heating is a step completed withina much shorter time than the drying step and the subsequent germinationinduction step. In other words, it is also a step in which intenseheating is conducted within a short time.

The effect of such a heating step is that germination is enhanced bysubjecting the dormant grape seeds to strong external stimulation. Thegrape seeds make a transition from the dormant state to the germinationand differentiation step.

Here, the inventors have conducted hydration of the grape seedssimultaneously with the heating step or after the heating step. This isbecause the heating caused the grape seeds to make a transition from thedormant state to the germination and differentiation state and the grapeseeds absorb a large amount of water as the germination anddifferentiation begin. It goes without saying that this does not excludea process of continuous hydration in the subsequent germinationinduction step.

The water content ratio increased by the germination and differentiationis preferably at least 17% or more. In other words, whether or not thegrape seeds have made a transition from the dormant state to thegermination and differentiation step can be checked by verifying thatthe water content ratio is 17% or more. In this case, if the seeds havenot been germinated and differentiated, they cannot be hydrated to awater content ratio of more than the aforementioned 14% even if immersedin water. A germination enhancement hormone can be added after theheating step.

Then, the inventors have conducted germination induction by curing thegrape seeds that have been sufficiently hydrated by curing in atemperature range optimum for germination. Germination or germinationand differentiation of the grape seeds is thereby enhanced. In thepresent invention, because the grape seeds passed through theabove-described steps, the germination ratio can be found to besignificantly increased by comparison with the conventional processes.Under certain conditions, a germination ratio of 95% and higher isobserved. From the standpoint of plant physiology, because of activeaction of this germination and differentiation, modification ofpolyphenols present in the grape seeds was confirmed. Presently about 30types of polyphenols present in the grape seeds have been identified,but in the present invention, main attention is focused on modificationof proanthocyanidins. Glycosidation of sugars following the germinationand differentiation of grape seeds results in the development ofpolarity, and a transformation is made from water-insolubleproanthocyanidins to water-soluble ones.

BEST MODE FOR CARRYING-OUT THE INVENTION

A method for the manufacture of polyphenols by using grape seeds as astarting material will be described below.

(Manufacturing Means)

First, an example of means for industrially extracting polyphenols thatwere made water soluble from grape seeds will be explained. Only oneexample of the apparatus used in each step is described, but thebelow-described apparatuses are not limiting, provided that theprocessing of each step can be executed.

Wine pomace and juice pomace are the starting materials that are theeasiest to be procured in large amounts. Those starting materials areused after allowing them to stay under conditions facilitating dryingover the prescribed period after squeezing out the juice, so that thewater content ratio thereof is decreased. If those starting materialsare not subjected even to the usual treatment, they have a very poorgermination ratio. In particular the germination ratio of the winepomace is poorer than that of the juice pomace, which is apparently dueto a germination inhibition action of ethyl alcohol.

(Sorting and Washing Step)

The object of this step is to obtain individually washed grape seeds.

Grape seeds and peels are sorted with a sorting apparatus equipped witha punching metal and then the classified grape seeds are stirred in awashing machine to remove contamination thoroughly.

(Heating Step)

The grape seeds obtained in the above-described sorting and washing stepare charged into a boiling kettle. They are heated for the prescribedtime at a prescribed temperature. As a result, the grape seeds emergefrom the dormant state.

(Germination Induction Step)

The grape seeds heated in the heating step are taken out and subjectedto germination induction in a curing kettle held at the prescribedpreferred germination temperature. Germination and differentiation ofthe grape seeds is initiated in the curing kettle and the water contentratio increases rapidly.

(Extraction Step)

Grape seeds whose germination or germination and differentiation haveadvanced in the above-described step are charged into a crushingapparatus, crushed, mixed with water and converted into an emulsion. Inthis step, a salt such as table salt or ammonium sulfate may be added toprecipitate (salt out) proteins and dissolve (salt in) water-insolublesubstances.

(Filtration Step)

Other components are further extracted from the precipitated layer withan organic solvent (ethyl alcohol and the like). After the water-solubleportion has been subjected to a known desalting treatment, water isevaporated with a vacuum apparatus and polyphenols comprisingproanthocyanidins converted to a water soluble form in a concentratedstate is obtained. Water-insoluble components are further extracted fromthe residue with an organic solvent.

The results obtained in simulating the process of obtainingwater-soluble polyphenols in accordance with the present invention willbe explained below as working examples.

WORKING EXAMPLE 1

1) Simulation Conditions

A total of 25 g of seeds were selected for use from grape seeds(Riesling type: water content 9.5-11.0) derived from juice pomace thatwas open-air stored for 6 months. After washing in running water, theseeds were immersed in 100 ppm antiformin solution (antiformin is abactericide) at 500 and incubated for 1 h. After 1 h, the seeds weretaken out from the solution, immersed into antiformin solution at 40°and incubated for 4 h. The seeds were then taken out of the solution andwashed with distilled water. Water was thereafter removed with softpaper. The seeds were then planted into a sponge containing 10 ppmantiformin solution and allowed to stay at 24° C. The temperature of 24°C. is the optimum germination temperature for grape seeds, but in orderto enhance water absorption of strong seeds, a germination preparationperiod of placing in a rather high-temperature zone such as 40° C. wasset prior to the optimum temperature. A 10 ppm antiformin solution wassprinkled for 12 h to prevent the seeds from drying.

2) Extraction of Germinated Seeds

At a timing of 24 h (1 day), 48 h (2 days), 96 h (4 days), and 168 h (7days), respectively, 100 seeds (4.12 g) were selected, 10 mL of buffersolution (distilled water) was added, and the seeds were crushed with amortar and a pestle. The product was centrifugally separated for 15 minat 10,000×g and the supernatant thereof was used as a sample.

3) Measurement of Water-Soluble Polyphenols

The absorbance at each wavelength of 220 nm, 280 nm, and 550 nm wasmeasured with a spectrophotometer with respect to the sample. At awavelength of 550 nm, measurements were conducted by coloring the sampleby adding 0.5 mL of 0.1% iron (III) chloride to 4 mL of the sampleliquid. At a wavelength of 220 nm, light absorption specific to aminoacids and proteins was observed. At a wavelength of 280 nm, lightabsorption specific to substances having a benzene ring was observed,and at a wavelength of 550 nm, light absorption specific to substancescomprising phenols was observed. If polyphenols are present, lightabsorption can be observed at both the wavelength of 280 nm and thewavelength of 550 nm.

The amount of polyphenols was determined by titration with a phenolreagent and taking gallic acid as a standard. Furthermore, the sampleswere obtained by using as starting materials the grape seeds sampledmultiple times from different zones of the juice pomace, and an averagevalue was taken for a total of ten measurements conducted by usingsimilarly obtained samples. The results are shown in Table 1.

Furthermore, the presence of polyphenols in the samples was confirmed bythe well-known Folin-Ciocalteu reaction. The presence of glycosides wasconfirmed by the well-known phenol-sulfuric acid method.

Table 1

4) Results

The absorbance at a wavelength of 220 nm reached a maximum in the firstday and then changed steadily with repeating moderate fluctuations. Inother words, the increase in amino acids and proteins was not confirmed.On the other hand, the absorbance at a wavelength of 280 nm increasedsignificantly with each passing day. In other words, it can be said thatthe amount of a substance comprising a benzene ring increased. Thissubstance is supposedly a polyphenol, but there remains a possibilitythat the amount of proteins or amino acids, which are other mainingredients, has increased. For this reason, the absorbance at awavelength of 550 nm was also measured. As a result, a significantincrease was also confirmed at a wavelength of 550 nm. Those results ledto a conclusion that the substance whose amount has increased is apolyphenol (there are also amino acids demonstrating light absorption atboth the wavelength of 280 nm and the wavelength of 550 nm, butgenerally they are absent and judging by the amounts, the possibility ofthem being present is zero).

Furthermore, as was confirmed by using thin-layer chromatography, adistinct reaction was obtained for substances comprising polyphenols andsugars. In other words, a glycoside polyphenol (water-soluble) wasconfirmed to be present in a comparatively large amount in the sample.Because proanthocyanidins are polyphenols that are most specific forgrape seeds and are present therein at a highest ratio, those glycosidepolyphenols can be supposed to comprise a large amount of glycosides ofproanthocyanidins.

COMPARATIVE EXAMPLE 1

1) Simulation Conditions

A total of 25 g of seeds were selected for use from grape seeds(Riesling type: water content 9.5-11.0) derived from juice pomace thatwas open-air stored for 6 months. After washing in running water, 100seeds (4.12 g) were selected, 10 mL of buffer solution (distilled water)was added, and the seeds were crushed with a mortar and a pestle. Theproduct was centrifugally separated for 15 min at 10,000×g and thesupernatant thereof was used as a sample. The grape seeds of ComparativeExample 1 were not subjected to special treatment for germination andwere used directly after washing.

2) Measurement of Water-Soluble Polyphenols

Similar to the above-described Working Example 1, absorbance at eachwavelength of 220 nm, 280 nm, and 550 nm was measured with aspectrophotometer.

Furthermore, the amount of polyphenols was determined by titration byusing a phenol reagent and taking gallic acid as a standard. Startingmaterials from different zones similar to those of Working Example 1were used, a total of ten measurements were conducted and an averagevalue was taken. The results are shown in Table 2.

3) Results

The amount of polyphenol obtained in Comparative Example 1 was abouthalf that obtained after one day for grape seeds of Working Example 1.This polyphenol was inherently soluble in water and showed no increase,as in Working Example 1.

As was confirmed by using thin-layer chromatography, only a slightreaction was obtained with respect to a substance comprising both thepolyphenol and sugars. This is because there was no synthesis ofglycoside polyphenol accompanying the germination and differentiation.In other words, though a polyphenol (water-soluble) was confirmed to bepresent in the sample, it was confirmed that almost none of thepolyphenol was present as a glycoside polyphenol.

Table 2

COMPARATIVE EXAMPLE 2 WORKING EXAMPLE 1

1) Simulation Conditions

The residue of the sample used in Working Example 1 was filtered andwater was removed. Then 10 mL of buffer solution (95% ethyl alcohol) wasadded and stirring was conducted for 8 h under heating. The product wascentrifugally separated for 15 min at 10,000×g and the supernatantthereof was used as a sample.

2) Measurement of Water-Soluble Polyphenols

Similarly to the above-described Working Example 1, absorbance at eachwavelength of 220 nm, 280 nm, and 550 nm was measured with aspectrophotometer.

Furthermore, the amount of polyphenols was determined by titration byusing a phenol reagent and taking gallic acid as a standard. An averagevalue was found for a total of ten measurements in the same manner as inWorking Example 1. The results are shown in Table 3.

3) Results

In Comparative Example 3, water-insoluble polyphenols were extracted.The amount of polyphenols in the first day was large. However, it wasconfirmed to decrease with each passing day. This phenomenon can beconsidered as glycosidation of water-insoluble polyphenols andconversion thereof into water-soluble glycoside polyphenols.

[Table 3]

TABLE 1 1 day 2 days 4 days 7 days Wavelength A220 0.583 0.291 0.3930.473 A280 0.292 0.452 1.41 0.982 A550 0.592 0.895 1.861 1.534 Amount ofpolyphenols 10.9 18.3 36.6 30.2 (mg/100 mL)

TABLE 2 Wavelength A220 0.303 A280 0.172 A550 0.311 Amount of 5.2polyphenols (mg/100 mL)

TABLE 3 1 day 2 days 4 days 7 days Wavelength A220 0.583 0.291 0.3930.473 A280 0.292 0.452 1.41 0.982 A550 0.592 0.895 1.861 1.534 Amount ofpolyphenols 10.9 18.3 36.6 30.2 (mg/100 mL) Amount of polyphenolsobtained 40.9 28.3 18.6 16.2 by ethanol extraction from residue (mg/100mL)

1. A method for obtaining water soluble polyphenols from grape seeds asa starting material, comprising: removing juice from the grape seeds;drying the grape seeds after removing the juice step; heating the grapeseeds after the drying step at a temperature range from 45° C. to 100°C.; hydrating the grape seeds with water; inducing germination of thegrape seeds, after the hydrating step by maintaining the grape seeds atemperature range optimum for germination, until the grape seedsgerminate; crushing the germinated grape seeds; mixing the crushedgerminated grape seeds with water and, evaporating the water after themixing step to form a concentrated extract from the germinated seedscontaining water soluble polyphenols.
 2. The method for obtaining watersoluble polyphenols, as recited in claim 1, wherein in the drying stepthe seeds are dried to a water content of 9 to 12 percent.
 3. The methodfor obtaining water soluble polyphenols, as recited in claim 1, whereinin the hydrating step the grape seeds are raised to a water contentratio of at least 17 percent.
 4. The method for obtaining water solublepolyphenols, as recited in claim 1, wherein the heating step has atemperature range from 50-70 degrees centigrade.
 5. The method forobtaining water soluble polyphenols, as recited in claim 1, wherein inthe drying step the grape seeds are dried to a water content of 9 to 11percent.
 6. The method for obtaining water soluble polyphenols, asrecited in claim 1, wherein the heating and hydrating steps areconducted simultaneously.
 7. The method for obtaining water solublepolyphenols, as recited in claim 1, wherein the hydrating step isconducted after the heating step.
 8. The method for obtaining watersoluble polyphenols, as recited in claim 1, wherein in the inducinggermination step the grape seeds are maintained at a temperature rangeof 23 to 40 degrees centigrade until germination occurs.
 9. The methodfor obtaining water soluble polyphenols, as recited in claim 1, whereinin the mixing step at least one of table salt and ammonium sulfate areadded to precipitate proteins and dissolve water-insoluble substances.10. The method for obtaining water soluble polyphenols, as recited inclaim 9, wherein the mixing step further includes a desalting treatmentafter the at least one of table salt and ammonium sulfate are added. 11.The method for obtaining water soluble polyphenois, as recited in claim1, wherein in the evaporating step, the concentrate is mixed with anorganic solvent to extract water insoluble components from theconcentrate.
 12. A method for extracting water soluble polyphenols fromdried grape seeds comprising: inducing germination of the grape seeds byheating and hydrating the grape seeds to awaken the grape seeds from adormant state and to increase the water content of the grape seeds to atleast 17 percent, wherein once the grape seeds are awakened, the grapeseeds are maintained at a temperature range optimum for germination,until the grape seeds germinate; and, extracting the water solublepolyphenols from the germinated grape seeds, including crushing thegerminated grape seeds, mixing the crushed grape seeds with water tocreate an emulsion and evaporating water from the emulsion.
 13. Themethod for extracting water soluble polyphenois from dried grape seeds,as recited in claim 12, wherein in the inducing germination step thegrape seeds are maintained at a temperature range of 23 to 40 degreescentigrade until germination occurs.